Investigating the diversity of bioluminescent marine worm Polycirrus (Annelida), with description of three new species from the Western Pacific

Bioluminescence, a phenomenon observed widely in organisms ranging from bacteria to metazoans, has a significant impact on the behaviour and ecology of organisms. Among bioluminescent organisms, Polycirrus, which has unique emission wavelengths, has received attention, and advanced studies such as RNA-Seq have been conducted, but they are limited to a few cases. In addition, accurate species identification is difficult due to lack of taxonomic organization. In this study, we conducted comprehensive taxonomic survey of Japanese Polycirrus based on multiple specimens from different locations and described as three new species: Polycirrus onibi sp. nov., P. ikeguchii sp. nov. and P. aoandon sp. nov. The three species can be distinguished from the known species based on the following characters: (i) arrangement of mid-ventral groove, (ii) arrangement of notochaetigerous segments, (iii) type of neurochaetae uncini, and (iv) arrangement of nephridial papillae. By linking the bioluminescence phenomenon with taxonomic knowledge, we established a foundation for future bioluminescent research development. We also provide a brief phylogenetic tree based on cytochrome c oxidase subunit I (COI) sequences to discuss the evolution of bioluminescence and the direction of future research.


Introduction
Bioluminescence is a well-documented phenomenon that occurs in many different organisms, including bacteria and fish, and has evolved independently in various lineages [1,2]. Bioluminescence has been reported in 14 annelid families, of which one is sipunculids [3], five are clitellates [4], and the rest are polychaetes [5][6][7]. Despite its occurrence, there is still much that is unknown about bioluminescence in polychaetes, including how it evolved and what it means ecologically. More research is needed to better understand this phenomenon, and to do this, we need to accumulate more examples of bioluminescent polychaetes [3,5,[7][8][9][10][11][12].
One genus of polychaetes that has been the subject of recent taxonomic research is Polycirrus Grube, 1850 [13] [14][15][16][17][18][19][20][21]. Unlike other genera of Terebelliformia, the members of Polycirrus do not make solid tubes, and they are found in shallow to deep waters, inhabiting environments such as sandy to muddy sediments, maerl or seagrass beds, the interior of sponges and the cracks of rocky or coral substrates [18,[21][22][23]. The classification of this genus and its 'family' has been the subject of much debate based on morphological and molecular data [21,[24][25][26]. Since this study does not focus on higher-level classification, we will treat Polycirrus as a genus of Terebelliformia without mentioning its family level. As Lavesque [21] has noted, future studies will probably shed more light on the taxonomy of this group. Genera similar to Polycirrus are known to include Amaeana Hartman, 1959 [27], Biremis Polloni, Rowe and Teal, 1973 [28], Enoplobranchus Verrill, 1879 [29], Hauchiella Levinsen, 1893 [30] and Lysilla Malmgren, 1866 [31] and can be differentiated based on the presence or absence of notochaetae and neurochaetae, and the form of the neurochaetae [22]. This paper adheres to the diagnosis of the genus Polycirrus according to Glasby & Hutchings [32]. Polycirrus currently consists of 77 described species from all over the world [18,21,32]. In Japan, two species have been described from this genus: P. nervosus Marenzeller, 1884 [33] from the coast of Enoshima and P. medius Hessle, 1917 [34] from the subtidal area of the Sagami Sea [33,34]. It is also known that members of this genus have the potential for bioluminescence, but only four species have been documented as bioluminescent to date: P. aurantiacus Grube, 1860 [35] (Croatia), P. perplexus Moore, 1923 [36] (USA), Polycirrus sp. (Hawaii), and Polycirrus sp. (Japan) [37][38][39][40]. Huber et al. [39] reported for Polycirrus perplexus a peak emission of 445 nm, noting it as an exceptionally short wavelength for a coastal organism. In line with this, Kanie et al. [40] have recently reported that one unnamed Japanese species of Polycirrus emitted light at a wavelength of 444 nm. Further research into the unique luminescence of this genus could provide valuable insights into the mechanisms and evolution of luminescent phenomena.
In this study, we describe three bioluminescent species of the genus Polycirrus that were obtained from various parts of Japan. After conducting a thorough examination of the specimens based on morphological and genetic information, we concluded that they did not match to any of the known species in the genus, including the two species previously described from Japan. We therefore describe them as three new species: Polycirrus aoandon sp. nov., Polycirrus ikeguchii sp. nov. and Polycirrus onibi sp. nov. We also provide the DNA barcode of the cytochrome c oxidase subunit I (COI) region for these species and present a concise molecular phylogenetic relationship tree of the three new species. Additionally, we have recorded videos of the luminescence activities exhibited by these species of Polycirrus.

Material and methods
Polycirrus worms were collected from various locations in Japan (figure 1) and examined for bioluminescence both in the field and in the laboratory [40]. The photographs and videos were taken royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230039 with a digital camera (α7S, Sony, Tokyo, Japan) with a macro lens SEL50M28 (Sony) for laboratory observation, and with SEL24F18Z lens (Sony) with an underwater camera housing (Nauticam NA A7, Nauticam, Hong Kong, China) for in situ observation. The bioluminescence was stimulated by poking with tweezers in the laboratory or by air bubbling from SCUBA gear in the field. The sampling was conducted with the permission of the local government (permission no.: i-1010). The specimens were preserved in 70% ethanol and subsequently examined under stereomicroscopes (Nikon SMZ1500 and Nikon Ni-U). The specimens were then deposited in the National Museum of Nature and Science in Tsukuba (NSMT). In this study, we followed the morphological terminology of Glasby & Hutchings [32] and Lavesque et al. [21].
To obtain sequences in the barcoding region of the mitochondrial cytochrome c oxidase subunit I (COI) gene, DNA extraction, sequencing, alignment and removal of ambiguous positions were carried out using the methods described in Jimi et al. [41]. DNA was extracted from the tentacles of the holotype, and the newly obtained sequences were deposited in GenBank (Accession nos. OQ067377-OQ067380 (https://www.ncbi.nlm.nih.gov/genbank)). In addition to these sequences, additional sequences for other Polycirrus species were obtained from GenBank. A total of 23 sequences (for 21 species) were used in the phylogenetic analysis by MEGAX based on 657 bp of COI using maximumlikelihood method with a GTR + G evolutionary model [42]. The LSID for this publication is: urn:lsid: zoobank.org:pub:2C00A2F7-7CA1-490C-A2CA-20CB85AA80B0.

Bioluminescent behaviour
At Sugashima Marine Biological Laboratory, we conducted an observation of the luminescent activity displayed by Polycirrus onibi sp. nov. and P. aoandon sp. nov. (figure 2a-d). Upon mechanical stimulation using tweezers, the tentacles of both species emitted an intense blue-purple luminescence. The stimulated tentacles flash (duration about 0.1 s) for a short period of time (0.3-1.1 s). The luminescent flashes were not found to trigger the neighbouring tentacles or to synchronize in an individual. Since we have only compared the luminescence of one individual from each species, we cannot draw definitive conclusions regarding the differences in the luminescence patterns between the two species. Electronic supplementary material, videos S1 and S2 provide further documentation of the luminescence of both species, and electronic supplementary material, video S3 has been provided for in situ observation of bioluminescence by SCUBA diving at Notojima (as depicted in figure 2e). The luminescence of Polycirrus in the colony was absent without bubbling stimulation, and it was evoked by bubbling air.     Diagnosis. Polycirrus with transparent body wall, tentacles with subterminal red spots, mid-ventral groove from segment 3, notochaetae on segments 3-14, neurochaetae on segment 15 and following segments, type II uncini, nephridial papillae in anterior area of parapodia on segments 3-14.
Description. Body wall transparent in life and whitish after fixation with ethanol (figures 2a and 3), slightly broader until segment 7, then gradually tapering to narrower uniformly cylindrical posterior body. Dorsum anteriorly tessellated. Venter anteriorly with mid-ventral groove and ventro-lateral pads (figure 3d ); pads tessellated, extending posteriorly from segment 3 to segment 13. Mid-ventral groove from segment 3.
Prostomium fused with base of upper lip and unclear boundary in dorsal and ventral sides. Buccal tentacles white in life and after fixation (figure 3c,d), two types: long and thin tentacles uniformly cylindrical; long and thick ones deeply grooved. Red spots present subterminally on both types of tentacles in life, faded after fixation with ethanol. Peristomium forming lips. Upper lip trefoiled with lateral blindly ending enclosed diverticula, margin of medial lobe convoluted; oral surface glandular, ciliated, with grooves leading to mouth. Inner lower lip oblong, smooth; outer region flat, shield-like, rounded and pointing toward mouth, ridged and grooved, extending posteriorly to segment 2. Achaetous segments visible dorsally but obscured by expanded outer lower lip ventrally.
Pygidium rounded, red pigmentation presents in life (figure 3a), faded after preservation. Etymology. The new species name derives from the Japanese yokai 'onibi'. Onibi represents the soul of a deceased human or animal, manifested as a floating blue flame. It is often equated with the Will-o'the-wisp. The blue-purple bioluminescence is reminiscent of this yokai.
Distribution and habitat. Only known from the collection sites, Sugashima (Mie, the northwestern Pacific Ocean) and Notojima (Ishikawa, the Sea of Japan); 1 m depth; muddy sediments or inside cracks of rocks.
Remarks. This species resembles Polycirrus disjunctus Hutchings & Glasby, 1986 [22] in having similar shape notopodial pre-and post-chaetal lobes, neurochaetae on segment 15 and type II uncini. The new species can be discriminated by the arrangement of notochaetae and nephridial papillae. While notochaetae are present on segments 3-14 and nephridial papillae on the anterior side of parapodia in P. onibi sp. nov., notochaetae present on segments 3-13 and nephridial papillae present on the posterior side of parapodia in P. disjunctus.
Two species, Polycirrus medius Hessle, 1917 [34] and P. nervosus Marenzeller, 1884 [33] have been described from Japan [32][33][34]. Polycirrus medius has been collected from Sagami Sea, approximately 30-40 m depth, in muddy sediment. The new species differs from P. medius by the presence of neurochaetae beginning about the last notochaetigerous segment (well before the last notochaetigerous segment in P. medius), by the presence of mid-ventral groove from segment 3 (from segment 4 or 5 in P. medius), by the presence of ventral pads on segments 3-13 (on segments 2-8 in P. medius), by rounded lower lip (oblong in P. medius), and by the presence of one type of notochaetae on segments 3-14 (two types of notochaetae on segments 3-17 in P. medius). Polycirrus nervosus has been collected from the east coast of Enoshima Island, intertidal, rocky shore. The new species differs from P. nervosus by the presence of ventral groove from segment 3 (segment 4 in P. nervosus), by the number of notochaetigerous segments (12 in P. onibi sp. nov., 42 in P. nervosus), by the absence of leafshaped tentacles ( present in P. nervosus), by the shape of upper lip (trefoiled in P. onibi sp. nov., convoluted in P. nervosus), by the presence of two types of notochaetae (one type in P. nervosus), and by the presence of type II uncini (type I uncini in P. nervosus). Upper lip trefoiled with lateral blindly ending enclosed diverticula, margin of medial lobe convoluted; oral surface glandular, ciliated, with grooves leading to mouth. Inner lower lip oblong, smooth; outer region flat, shield-like, rounded and pointing toward mouth, ridged and grooved, extending posteriorly to segment 2. Achaetous segments visible dorsally but obscured by expanded outer lower lip ventrally.
Etymology. The species is named after Mr Shinichiro Ikeguchi. He is the former deputy director of the Notojima Aquarium, and he contributed to the discovery of the luminescence phenomenon of this species.
Distribution and habitat. Only known from the collection sites, off Shirawara Coast and off Cape Haneo (Tottori, the Sea of Japan) and Notojima (Ishikawa, the Sea of Japan); 1-15 m depth; muddy sediments or inside cracks of rocks.
Remarks. This species resembles Polycirrus aquila Caullery, 1944 [43] and P. coccineus Grube, 1870 [44] in the type of notochaetae and neurochaetae, having pinnate notochaetae and type II uncini. The new species differs from P. aquila by the presence of two types of notochaetae (one type in P. aquila) and by the presence of notochaetae and nephridial papillae on segments 3-19 (on segments 3-16 in P. aquila). The new species differs from P. coccineus by the presence of notochaetae on segments 3-16 (on Polycirrus ikeguchii sp. nov. can be discriminated from other known species of Japan (P. medius, P. nervosus and P. onibi sp. nov.) by the number of notochaetigerous segments (17 in P. ikeguchii sp. nov., 15 in P. medius, 42 in P. nervosus, 12 in P. onibi sp. nov.), by the presence of nephridial papillae on segments 3-19 (3-8 in P. medius, 3-10 in P. nervosus, 3-14 in P. onibi sp. nov.), neurochaetae beginning around the last notochaetigerous segment (well before the last notochaetigerous segment in P. nervosus, around the last notochaetigerous segment in P. medius and P. onibi sp. nov.), and type II uncini (type I in P. nervosus, type II in P. medius and P. onibi sp. nov.). DNA barcode (Genbank No. LC601006) and RNA-seq data (Genbank No. DRX256761, DRX256762) provided in Kanie et al. [40] were based on this species. royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230039 absent, two types: long and thin tentacles uniformly cylindrical; long and thick ones deeply grooved. Peristomium forming lips. Upper lip trefoiled with lateral blindly ending enclosed diverticula, margin of medial lobe convoluted; oral surface glandular, ciliated, with grooves leading to mouth. Inner lower lip oblong, smooth; outer region flat, shield-like, rectangular, ridged and grooved, extending posteriorly to segment 2. Achaetous segments visible dorsally but obscured by expanded outer lower lip ventrally. Notopodia from segment 3, ending in segment 56; distinctly elongate, rectangular, first pair slightly shorter, bilobed, prechaetal and postchaetal lobes same shape, triangular. Notochaetae one type, smooth (figure 8a,c). Neuropodia beginning from segment 16; uncini with short neck and straight base (Type I) (figure 8b), teeth above main fang arranged in one transverse series (MF:3-5) (figure 8d ), enlarged median tooth above main fang present, subrostral process absent.
Pygidium rounded, pigmentation unknown in life and absent after fixation with ethanol.
Etymology. The new species name derives from the Japanese yokai 'Aoandon'. Aoandon carries a blue (= Ao in Japanese) lantern (= andon in Japanese). The blue-purple bioluminescence is reminiscent of this yokai.
Distribution and habitat. Only known from the type locality, Sugashima (Mie, the northwestern Pacific Ocean); 1-2 m depth; muddy sediments under rocks.

Discussion
Research on the genus Polycirrus has been on the rise in recent years, with 27 species being described since the year 2000. The increased interest in taxonomic research of this genus can be attributed to the global review conducted by Glasby and Hutchings in [32]. However, there has been a lack of taxonomic studies on Polycirrus in Japan since the description of P. nervosus by Marenzeller [33] and P. medius by Hessle [34]. This study represents a significant step forward in the study of Polycirrus diversity in Japan, particularly with the discovery of the new species P. onibi sp. nov., which is found in the shallow subtidal zone of the Sea of Japan (Notojima) and the North Western Pacific Ocean (Sugashima), and is therefore easily accessible for collection in ecological and other studies. Phylogenetic molecular analysis has confirmed that the three species described in this study are contained within the genus Polycirrus ( figure 9). However, the overall bootstrap support for this phylogenetic tree is low. Further sampling of operational taxonomic units is necessary to determine the evolution of these species. It has also been revealed that P. onibi sp. nov. and P. ikeguchii sp. nov., which have morphological similarities, are closely related. It is possible that the type of uncini (type II) is a synapomorphy reflecting their phylogenetic relationships.  Figure 9. Maximum-likelihood phylogenetic tree of Polycirrus species based on partial COI sequences in the barcoding region. Amaeana sp. and A. trilobata were used as an outgroup. Nodal supports (bootstrap support value in % from 1000 replicates) higher than 50% are indicated on each branch. Blue circles indicate species which have bioluminescent ability. GenBank accession numbers follow the species names.
royalsocietypublishing.org/journal/rsos R. Soc. Open Sci. 10: 230039 The ability to emit light occurs in various types of annelids, and it is thought to have been independently acquired multiple times [6]. The luminescence of Terebelliformia, the suborder to which Polycirrus belongs, has been poorly studied, with only a few reports on the genera Thelepus and Polycirrus [8,[37][38][39][40]. In this study, we compare the luminescence between two species of the same genus for the first time and find no apparent differences in their luminescence patterns or colours. Molecular analysis revealed that P. aoandon sp. nov. and P. onibi sp. nov. are not closely related, but their shared trait of luminescence suggests the possibility that luminescence is a common trait within the entire genus. However, since we only compared individual specimens, it is not possible to draw a definitive conclusion. It will be possible to elucidate whether there are differences in bioluminescence between different species by conducting further additional experiments in the future. Further research will help us better understand the phenomenon of bioluminescence in Polycirrus, which is known for its characteristic short-wavelength luminescence. Bioluminescence has also been observed in other genera in Terebelliformia, such as Thelepus, but the wavelength of this luminescence (508 nm) differs from that of Polycirrus (444 nm) [8,40]. In order to understand how bioluminescence has been acquired within Terebelliformia, further study on closely related taxa to Polycirrus, such as Amaeana, is necessary.
The ecological significance of bioluminescence in Polycirrus has been discussed in previous studies [39,40], with some suggesting that it may function as a warning to predators. The species discovered in this study are also known to live buried in mud or in crevices of rocks. Therefore, we opined that they cannot effectively communicate with their surroundings through luminescence unless they are dug up to expose themselves. This indicates that the luminescence of this genus may only be useful in times of emergency situations when their bodies are exposed. In our observations, we found that the organism emitted light when stimulated with a pinprick (figure 2, electronic supplementary material, videos S1 and S2). This suggests that the Polycirrus worm is emitting light in response to external stimuli, which supports the hypothesis that it is exhibiting a defensive response to potential threats.
Ethics. The sampling was conducted with the permission of the local government ( permission number: i-1010). Data accessibility. The videos depicting observation of bioluminescence are appended as appendix videos [46]. Genetic data can be obtained from Genbank (Accession nos. OQ067377-OQ067380: https://www.ncbi.nlm.nih.gov/genbank).